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Changes in S-(2-succinyl)cysteine and advanced glycation end-products levels in mouse tissues associated with aging


Cysteine is non-enzymatically modified by fumarate, which is an intermediate of the tricarboxylic acid cycle, leading to the formation of S-(2-succinyl)cysteine (2SC). Post-translational modification of physiological proteins by fumarate causes enzyme dysfunction. The aim of the study was to evaluate the changes in 2SC accumulation in physiological tissues associated with aging. Brain, liver, kidney, and serum samples were collected from 4-, 12-, and 96-week-old male C57BL/6J mice, and the level of 2SC was determined by liquid chromatography–tandem mass spectrometry (LC–MS/MS) after pretreatment, including delipidation, protein precipitation, and hydrolysis using hydrochloric acid. The 2SC level in the brain was higher than that in other tissues, and its accumulation significantly increased with age. Similarly, Nε-(carboxymethyl)lysine levels, an advanced glycation end-products (AGEs) that accumulates in tissues in an age-dependent manner, was found to be increased in the brain and kidneys of elderly mice. Accumulation of Nδ-(5-hydro-5-methyl-4-imidazolone-2-yl)-ornithine increased significantly with age, but only in the kidneys. The fumarate content in the brain was similar to that in the liver and kidney at 4 and 12 weeks of age. Furthermore, fumarate contents increased in the liver and kidney at 96 weeks of age, whereas its level did not change in the brain. Our results demonstrated that the changes in 2SC and AGEs levels in tissues reflected differing metabolism and enhanced oxidative stress in each organ; in particular, the metabolism in the brain and kidneys is highly affected by aging.

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Advanced glycation end-products


Bicinchoninic acid






Glyoxalase 1


Hydrochloric acid


Liquid chromatography–tandem mass spectrometry






Reduced nicotinamide adenine dinucleotide

NAD+ :

Oxidized nicotinamide adenine dinucleotide


Sodium hydroxide


Post-translational modification


Standard deviation


Trichloroacetic acid




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We are grateful to Jun-ichi Shirakawa and Hikari Satou from our laboratory for discussion about pretreatment of samples before analysis. The authors also thank Editage for editing the manuscript and providing suggestions.


This work was supported by a JSPS KAKENHI grant (grant no. 20K05895) provided to Ryoji Nagai. This work was also supported in part by the Japan Science and Technology Agency, Adaptable and Seamless Technology Transfer Program through Target-Driven R and D (number AS3015118U).

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NK: Master of agriculture made principal contributions to study design and to the acquisition, analysis, and interpretation of results. She wrote the greater part of this article. HT: took part in animal experiments. MN: took part in the acquisition of data by measuring 2SC and AGEs. HS: took part in animal experiments, acquisition of data, and interpretation of results. RN: contributed to study design, measuring 2SC and AGEs, and interpretation of results.

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Correspondence to Ryoji Nagai.

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The authors declare that they have no conflicts of interest with the contents of this article.

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Animal experiments were performed with the approval of the ethics review committee of Tokai University for animal experimentation (approval number 211019).

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Supplementary file1 (TIF 94 KB) A scheme of pretreatment procedures for the measurement of 2SC and AGEs. (A) A scheme of pretreatment for measurement of 2SC levels in tissues. (B) A scheme of pretreatment of tissues for measurement of AGEs levels.


Supplementary file2 (TIF 141 KB) Changes in AGEs levels normalized by protein. CML levels in the (A) brain, (B) liver, and (C) kidneys was measured by LC-MS/MS and normalized to the protein content of each tissue. MG-H1 levels in the (D) brain, (E) liver, and (F) kidneys was measured by LC-MS/MS and normalized to the protein content of each tissue. Comparison of AGEs contents at 4, 12, and 96 weeks of age is presented. Graph that shows the data as jittered dots. The summary of the data is shown as horizontal line indicating the median (n=6, ∗p < 0.05).

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Katsuta, N., Takahashi, H., Nagai, M. et al. Changes in S-(2-succinyl)cysteine and advanced glycation end-products levels in mouse tissues associated with aging. Amino Acids 54, 653–661 (2022).

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  • S-(2-succinyl)cysteine
  • Advanced glycation end-products
  • Aging-related changes
  • Liquid chromatography–tandem mass spectrometry
  • Brain metabolite levels